This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The leading work of Dr. Dean Li found that Robo4 plays a key role in stabilizing vascular networks by interacting with both Slit2 and Slit3. In vitro studies observed that treating Robo4-expressing human umbilical vein ECs with a soluble extracellular domain of Robo4 blocked Slit2 function and inhibited EC proliferation and migration, further supporting that Robo4 promotes angiogenesis. These studies have established the critical proangiogenic role of Slit-Robo4 interaction in angiogenesis. The production of structurally characterized HS and CS and the development of mutant endothelial cell lines, combined with cell-surface ligand binding and endothelial cell function assays, can greatly advance our knowledge and understanding of the regulatory role of HSPGs in Robo4 function and signaling. The combination of Resource recombinant protein expression, and the in vitro screening and synthesis of high affinity ligands will provide a platform for the rational design of glycosaminoglycan agents for modulation of Robo4 function in angiogenesis.